Characterization of mutations in crucial residues around the Q_o binding site of the cytochrome bc₁ complex from Paracoccus denitrificans

Abstract

The protonation state of residues around the Qo binding site of the cytochrome bc1 complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site‐directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme bL edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild‐type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Qo site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P. denitrificans as well. The protonated residue E295 is proposed to change the hydrogen‐bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X‐ray structures.

Zinc Inhibition of Bacterial Cytochrome bc1 Reveals the Role of Cytochrome b E295 in Proton Release at the Qo Site

“…The spectral region between 1750 and 1700 cm −1 includes information about the protonated Asp/Glu residues, and can be used as a good indicator of the environment of these redox active amino acids. The oxidation-induced protonation of acidic residues gives rise to a positive signal at 1739 cm −1 , which was previously assigned to the υ(C=O) vibration of D278 and E295 residues in cyt bc 1 (26, 35, 36, 38, 41). The negative signal observed at 1720 cm −1 can be assigned to the υ(C=O) vibration of protonated acidic residues.…”

“…The observation that yeast E272V or bacterial E295V mutants did not show any significant effect on the Q i site-mediated reverse electron transfer rate (37) or the Q o site-mediated cyt b H reduction rates (26), respectively, suggests that no alteration of the electron transfer pathway is caused by the E295V mutation. In addition, the physicochemical properties of cyt bc 1 ( i.e ., E m,7 of hemes b L and b H or redox sensitive spectra) were not altered by mutation of E295 in bacterial cyt bc 1 (26, 38). On the other hand, the rate of b H reduction of E295Q at different pH values in bacterial (26), as well as the turnover rate of cyt c reduction in E272D and Q at below pH 6 in yeast (36) mutants were significantly decreased as compared to those of the corresponding wild-type enzymes.…”

“…A positive signal can be seen at 1706 cm − 1 . This signal was tentatively assigned to an acidic residue located in the heme b subunit of P. denitrificans (38, 43). Furthermore, this signal can arise also from the υ(C=O) vibration of the heme propionates.…”

“…4). The redox-induced FTIR difference spectra provide information on the protonation state of acidic residues or quinone binding, as described previously (35, 38, 40, 41). The positive and negative signals in the spectra correlate with the oxidized, and the reduced forms of the enzyme, respectively.…”

Infrared spectroscopic markers of quinones in proteins from the respiratory chain

“…It is possibly at the origin of the shifts seen here. The contribution of residues from the Qo binding site to this spectral range and their protonation state was further probed in the bc 1 complex from P. denitrificans [81]. On the basis of the comparison of the electrochemically induced FTIR difference spectra of the wild type (Fig.…”